The present invention relates to resistance genes, DNA constructs, micro organisms, plant cells and plants comprising said resistance genes. Furthermore the invention relates to genetically transformed plants which are resistant against nematodes and/or aphids. In addition, the invention relates to probes, and primers for the identification of the resistance genes and diagnostic kits comprising said probes and/or primers. Finally, the invention relates to polypeptides encoded by said resistance genes and the use of said polypeptides.
Plant pathogens are responsible for substantially losses of plants and plant products due to infection of the plant. Plant diseases, as a result of infection by plant pathogens or pests, cause damage to the plants and/or plant products, reduce production and yield, limit the Kind of plants that can grow in certain geographic areas and as a result cause severe (financial) losses to the grower.
Plant parasitic nematodes occur worldwide and most of them live most of their life in the topsoil layer. Although losses caused by direct feeding of nematodes on plant roots is considered to be of minor importance, several species, among them the root-knot nematodes belonging to the Meloidogyne species, the cyst nematodes belonging to the Heterodera species and Globodera species and other nematodes such as the Nacobbus species, cause severe damage and economic crop losses. Root-knot nematodes also occur throughout the world but are found more frequently and in greater numbers in areas with warmer climates and in greenhouses. The most important Meloidogyne species are M. incognita, M. arenaria, M. hapla and M. javanica, of which M. hapla also occurs in more temperate climatic zones.
Different means for control of the plant pathogens exist, such as mechanical cultivation of the soil, chemical treatment with pesticides, including nematicides and insecticides, or crop rotation. However, for certain plant pathogens, especially nematodes, these means of control are insufficient to protect the plants from infection and resulting diseases. The only effective means of control involves plant host resistance (Russell, 1978, Plant Breeding for pest and disease resistance, Butterworths edit., 485 pp). The development of cultivars resistant to common plant pathogens is one of the major goals of plant breeders today, in order to reduce or ultimately eliminate the extensive need for pesticides. The burden for the environment of the large amounts of pesticides injected into the soil or sprayed on crops, trees etc. worldwide each year becomes too severe. Moreover, governmental regulations in Western countries restrict the use or even forbid the use of certain pesticides. Therefore, the need for plants which are resistant to one or more of their pathogens, or which have a reduced susceptibility to their attackers becomes more and more pressing. The development of resistant plants is one of the important objectives of current plant breeding programs. Plant genotypes susceptible for particular pathogens are crossed with resistant plant genotypes in order to introduce the resistant phenotype into the breeding line.
Damage by root-knot nematodes results primarily from the invasion of the plant roots by larvae which in a compatible relationship with the plant develop into a reproducing female. After invasion the larvae cause root cells to develop into giant cells on which they feed. Upon infection galls or knots are formed on the roots and the plant roots become otherwise disturbed, thickened and stunted. The root system thus disfunctions in the uptake of water and nutritional elements which damages the plant growth and development. Frequently damage to infected plants is increased by parasitic fungi attacking the weakened root tissue. Infected plants show reduced growth and smaller pale coloured leaves, with dwarf poor quality fruits or even without fruits, and tend to wilt in warmer climates (Agrios, 1988 in: Plant Pathology, Academic Press, Inc.). The damage and/or yield reduction caused by root-knot nematodes is substantial on the total agricultural production worldwide. In individual stand yield losses can be as high as 25-50%, or even a crop may be killed.
In greenhouses root-not nematodes can be controlled with steam sterilization of the soil or soil fumigation with nematicides. Under field conditions control can be achieved by the use of nematicides. However, the use of such, in some cases very persistent, chemicals is increasingly debated and in some countries the use of certain nematicides is even forbidden.
Breeding genetically resistant genotypes is the most reliable and effective way of controlling root-knot disease. For a number of crop species the availability of resistance within the related germplasm has been reported, e.g. potato, cotton, tobacco, wheat, soybean, tomato, eggplant, common bean and alfalfa. Resistance breeding is hampered by firstly the limited occurrence of (known) resistance genes in the available germplasm, secondly, in some plant species the existence of crossing barriers between the cultivated crop species and the resistance bearing related species, and thirdly, screening tests for resistance versus susceptibility to nematodes are laborious and often not reliable. Therefore, resistance breeding is very difficult or not to achieve, or if possible time consuming.
Successful introduction of resistance genes has been realized in tomato. The resistance gene Mi (Meloidogyne incognita) has been introduced into cultivated tomato, Lycopersicon esculentum, after crossing with the related wild species L. peruvianum (PI 128657), using embryo culture. The Mi gene confers resistance to various Meloidogyne spp. (Fassuliotis, 1991, in: Genetic Improvement of Tomato, Springer Verlag edit.). The Mi resistance gene is reported to be a monogenic dominant gene (Gilbert and McGuire, 1956, Proc. Am. Soc. Hortic. Sci. 68, 437-442) and is located on tomato chromosome 6. It is also postulated that the introgressed region comprising the Mi locus is involved in conferring resistance to potato aphid (Macrosiphum euphorbia) (Kaloshian et al, 1995, Proc. Natl. Acad. Sci. USA, 92, 622-625).
Plants have developed a complex defense mechanism against attack and infection by pathogens. To date, the exact mechanism of their defense system is not yet elucidated.
Nematode resistance in tomato is expressed after penetration. After the juvenile larva enters the root and establishes itself at a feeding site, a hypersensitive reaction (HR) adjacent to the head of the nematode is triggered that results in local death of the host cells. The nematode is also adversely affected by this HR and dies (Fassuliotis, 1991, in: Genetic Improvement of Tomato, Springer Verlag edit.). Wether or not there exists a gene-for-gene relationship sensu Flor (1956, Adv.Gen. 8, 29-54) as is frequently the case in other plant-pathogen relationships where resistance is based on HR-incompatibility is unknown.
The isolation of plant genes without knowing their gene products is very laborious and difficult, because of the enormous genome sizes of plant species: e.g. tomato has a genome size of 1000 Mb (109 base pairs of nuclear DNA), maize has a genome size of 3000 Mb and wheat has even more than 16xc3x97109 base pairs. Searching for a specific gene among these billions of base pairs is only feasible when (i) there are enough molecular markers tightly linked to the gene of interest and (ii) there is good genetic material available (Tanksley et al., 1995, Trends in Genetics, 11, p. 63-68).
Although, the isolation of a few resistance genes has been reported, none of these resistance genes are able to confer the host plant resistant to nematodes or to aphids. Examples of such isolated resistance genes are: RPS2 from Arabidopsis (resistance to Pseudomonas syringae expressing avrRpt2), N from tobacco (resistance to tobacco mosaic virus), Cf-9 from tomato (resistance to the leaf fungal pathogen Cladosponrum fulvum carrying avr9) and L6 from flax (resistance to the corresponding leaf rust fungal race) (Dangl, 1995, Cell 80, 363-366).
The present invention provides the first isolated nematode resistance gene, and furthermore, provides the first isolated aphid resistance gene. Moreover, the present invention relates to a dual function resistance gene conferring reduced susceptibility to nematodes as well as aphids, and preferably to Meloidogyne incognita and Macrosiphum euphorbiae respectively.
The present invention relates to a nucleic acid comprising the Mi resistance gene which when present and expressed in a plant is capable of conferring to said plant resistance against nematodes and/or aphids. Furthermore, the invention relates to the Mi resistance gene of which the DNA sequence is disclosed herein. The invention also relates to a gene product encoded by the Mi resistance gene. In addition, the present invention relates to DNA constructs, cosmids, vectors, bacterial strains, yeast cells and plant cells comprising the Mi resistance gene. In another aspect, the present invention relates to a genetically transformed plant, which is resistant to a nematode, said nematode being capable of infecting the untransformed plant. Furthermore, the invention relates to resistance genes which are homologous to the Mi resistance gene, and which, when present in a plant, are able of conferring said plant resistance to infection by pathogens.
Moreover, the present invention relates to a nucleic acid comprising the Meu-1 resistance gene which when present in a plant is capable of conferring to said plant reduced susceptibility to aphids. In particular the Meu-1 resistance gene corresponds to the Mi resistance gene. Especially the Meu-1 resistance gene has the same nucleotide sequence as the Mi resistance gene. Thus, the present invention also relates to genetically transformed plants, which are reduced susceptible, and preferably resistant to aphids, in particular to potato aphids.
Finally, the invention relates to oligonucleotides corresponding to the sequence of the said resistance gene or part thereof, and detection kits comprising said oligonucleotides.